A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism

ALS8 is caused by a dominant mutation in an evolutionarily conserved protein, VAPB (vesicle-associated membrane protein (VAMP)-associated membrane protein B)/ALS8). We have established a fly model of ALS8 using the corresponding mutation in Drosophila VAPB (dVAP33A) and examined the effects of this mutation on VAP function using genetic and morphological analyses. By simultaneously assessing the effects of VAP(wt) and VAP(P58S) on synaptic morphology and structure, we demonstrate that the phenotypes produced by neuronal expression of VAP(P58S) resemble VAP loss of function mutants and are opposite those of VAP overexpression, suggesting that VAP(P58S) may function as a dominant negative. This is brought about by aggregation of VAP(P58S) and recruitment of wild type VAP into these aggregates. Importantly, we also demonstrate that the ALS8 mutation in dVAP33A interferes with BMP signaling pathways at the neuromuscular junction, identifying a new mechanism underlying pathogenesis of ALS8. Furthermore, we show that mutant dVAP33A can serve as a powerful tool to identify genetic modifiers of VAPB. This new fly model of ALS, with its robust pathological phenotypes, should for the first time allow the power of unbiased screens in Drosophila to be applied to study of motor neuron diseases.     

From ER to Eph receptors: new roles for VAP fragments

Dominantly inherited mutations in an endoplasmic reticulum protein called VAPB have been found in a subset of patients with a rare familial form of amyotrophic lateral sclerosis (ALS). In this issue, Tsuda et al. (2008) identify a secreted form of VAPB that binds directly to Eph receptors inducing their activation and signaling, providing fresh insights into ALS pathogenesis, including non-neuronal aspects of this disorder.     

Novel splice variants of the amyotrophic lateral sclerosis-associated gene VAPB expressed in human tissues

VAPB is a highly conserved integral membrane protein that is ubiquitously expressed in all eukaryotic organisms and located within the membranes of the endoplasmic reticulum (ER). The P56S missense mutation of the VAPB protein is linked to a hereditary form of amyotrophic lateral sclerosis (ALS8), and the pathogenesis of ALS8 has remained enigmatic. We report the cloning of five novel splice variants of the human VAPB gene, all of which are expressed at the mRNA level in the human nervous system. When transfected into human HEK293 or SH-SY5Y cells, two of these variants (VAPB-2 and VAPB-4,5) were readily detectable by immunoblotting whereas two variants (VAPB-3 and VAPB-3,4) became detectable after proteasomal inhibition, a condition commonly found in neurodegenerative diseases. Interestingly, one of these novel VAPB variants, VAPB-2, co-immunoprecipitated with wt-VAPB. However, so far none of these splice variants could be detected by immunoblotting of lysates from selected human tissues, suggesting that in vivo, the proteins translated from the variant VAPB mRNAs are quickly degraded or, alternatively, the expressed proteins are below detection limit of the available antibodies. We speculate that under conditions of proteasomal inhibition, as encountered in many neurodegenerative diseases including ALS, variant VAPB proteins might accumulate in affected cells and contribute to ALS pathogenesis.     

Structural, stability, dynamic and binding properties of the ALS-causing T46I mutant of the hVAPB MSP domain as revealed by NMR and MD simulations

T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.     

Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8)

The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8.     

Secreted VAPB/ALS8 major sperm protein domains modulate mitochondrial localization and morphology via growth cone guidance receptors

The VAPB/ALS8 major sperm protein domain (vMSP) is implicated in amyotrophic lateral sclerosis and spinal muscular atrophy, yet its function in the nervous system is not well understood. In Caenorhabditis elegans and Drosophila, the vMSP is cleaved from its transmembrane anchor and secreted in a cell type-specific fashion. We show that vMSPs secreted by neurons act on Lar-like protein-tyrosine phosphatase and Roundabout growth cone guidance receptors expressed in striated muscle. This signaling pathway promotes Arp2/3-dependent actin remodeling and mitochondrial localization to actin-rich muscle I-bands. C. elegans VAPB mutants have mitochondrial localization, morphology, mobility, and fission/fusion defects that are suppressed by Lar-like receptor or Arp2/3 inactivation. Hence, growth cone guidance receptor pathways that remodel the actin cytoskeleton have unanticipated effects on mitochondrial dynamics. We propose that neurons secrete vMSPs to promote striated muscle energy production and metabolism, in part through the regulation of mitochondrial localization and function. VIDEO ABSTRACT:     

Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S)

A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAP Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAP activities.     

Amyotrophic lateral sclerosis-linked mutant VAPB enhances TDP-43-induced motor neuronal toxicity

Transactive response DNA-binding protein-43 (TDP-43) has been thought to be generally involved in the pathogenesis of most amyotrophic lateral sclerosis (ALS) patients although it remains undefined how TDP-43 is involved in the ALS pathogenesis. In this study, we found that a P56S mutant of vesicle-associated membrane protein-associated protein B (VAPB), which has been identified to be a familial ALS-causative protein, potentiated the TDP-43-induced motor neuronal cell death, while wild-type VAPB conversely inhibited it. The P56S-VAPB-induced potentiation of the TDP-43-induced death was mediated by the up-regulation of Bim expression at the mRNA level and other undefined mechanisms that leads to the enhancement of Bim and Bax activity. These observations suggest that TDP-43 and P56S-VAPB may co-operate to involve the pathogenesis of ALS.     

Intrinsically unstructured domain 3 of hepatitis C Virus NS5A forms a "fuzzy complex" with VAPB-MSP domain which carries ALS-causing mutations

Hepatitis C virus (HCV) affects nearly 200 million people worldwide and is a leading factor for serious chronic liver diseases. For replicating HCV genome, the membrane-associated replication machinery needs to be formed by both HCV non-structural proteins including NS5A and human host factors. Recently NS5A has been identified to bind ER-anchored human VAP proteins and consequently this interaction may serve as a novel target for design of anti-HCV drugs. So far no biophysical characterization of this interaction has been reported. Here, we dissected the 243-residue VAPB into 4 and 447-residue NS5A into 10 fragments, followed by CD and NMR characterization of their structural properties. Subsequently, binding interactions between these fragments have been extensively assessed by NMR HSQC titration which is very powerful in detecting even very weak binding. The studies lead to three important findings: 1). a "fuzzy complex" is formed between the intrinsically-unstructured third domain (D3) of NS5A and the well-structured MSP domain of VAPB, with an average dissociation constant (Kd) of ~5 μM. 2). The binding-important residues on both NS5A-D3 and VAPB-MSP have been successfully mapped out, which provided experimental constraints for constructing the complex structure. In the complex, unstructured D3 binds to three surface pockets on one side of the MSP structure. Interestingly, two ALS-causing mutations T46I and P56S are also located on the D3-MSP interface. Moreover, NS5A-D3, FFAT-containing proteins and EphA4 appear to have overlapped binding interfaces on the MSP domain. 3). NS5A-D3 has been experimentally confirmed to competes with EphA4 in binding to the MSP domain, and T46I mutation of MSP dramatically abolishes its binding ability to D3. Our study not only provides essential foundation for further deciphering structure and function of the HCV replication machinery, but may also shed light on rationalizing a recent observation that a chronic HCV patient surprisingly developed ALS-like syndrome.     

CYP2E1 and Parkinson’s disease in a MPTP-induced C57BL/6 mouse model

Background

A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca²⁺ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.

Results

To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.

Conclusion

Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.     

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.     

ER Stress and Unfolded Protein Response in Amyotrophic Lateral Sclerosis

Several theories on the pathomechanism of amyotrophic lateral sclerosis (ALS) have been proposed: misfolded protein aggregates, mitochondrial dysfunction, increased glutamate toxicity, increased oxidative stress, disturbance of intracellular trafficking, and so on. In parallel, a number of drugs that have been developed to alleviate the putative key pathomechanism of ALS have been under clinical trials. Unfortunately, however, almost all studies have finished unsuccessfully. This fact indicates that the key ALS pathomechanism still remains a tough enigma. Recent studies with autopsied ALS patients and studies using mutant SOD1 (mSOD1) transgenic mice have suggested that endoplasmic reticulum (ER) stress-related toxicity may be a relevant ALS pathomechanism. Levels of ER stress-related proteins were upregulated in motor neurons in the spinal cords of ALS patients. It was also shown that mSOD1, translocated to the ER, caused ER stress in neurons in the spinal cord of mSOD1 transgenic mice. We recently reported that the newly identified ALS-causative gene, vesicle-associated membrane protein-associated protein B (VAPB), plays a pivotal role in unfolded protein response (UPR), a physiological reaction against ER stress. The ALS-linked P56S mutation in VAPB nullifies the function of VAPB, resulting in motoneuronal vulnerability to ER stress. In this review, we summarize recent advances in research on the ALS pathomechanism especially addressing the putative involvement of ER stress and UPR dysfunction.     

The ALS8 protein VAPB interacts with the ER–Golgi recycling protein YIF1A and regulates membrane delivery into dendrites

The vesicle‐associated membrane protein (VAMP) associated protein B (VAPB) is an integral membrane protein localized to the endoplasmic reticulum (ER). The P56S mutation in VAPB has been linked to motor neuron degeneration in amyotrophic lateral sclerosis type 8 (ALS8) and forms ER‐like inclusions in various model systems. However, the role of wild‐type and mutant VAPB in neurons is poorly understood. Here, we identified Yip1‐interacting factor homologue A (YIF1A) as a new VAPB binding partner and important component in the early secretory pathway. YIF1A interacts with VAPB via its transmembrane regions, recycles between the ER and Golgi and is mainly localized to the ER–Golgi intermediate compartments (ERGICs) in rat hippocampal neurons. VAPB strongly affects the distribution of YIF1A and is required for intracellular membrane trafficking into dendrites and normal dendritic morphology. When VAPB‐P56S is present, YIF1A is recruited to the VAPB‐P56S clusters and loses its ERGIC localization. These data suggest that both VAPB and YIF1A are important for ER‐to‐Golgi transport and that missorting of YIF1A may contribute to VAPB‐associated motor neuron disease.     

First evidence of pathogenicity of V234I mutation of hVAPB found in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases.     

ALS-linked P56S-VAPB, an aggregated loss-of-function mutant of VAPB, predisposes motor neurons to ER stress-related death by inducing aggregation of co-expressed wild-type VAPB

A point mutation (P56S) in the vapb gene encoding an endoplasmic reticulum (ER)-integrated membrane protein [vesicle-associated membrane protein-associated protein B (VAPB)] causes autosomal-dominant amyotrophic lateral sclerosis. In our earlier study, we showed that VAPB may be involved in the IRE1/XBP1 signaling of the unfolded protein response, an ER reaction to inhibit accumulation of unfolded/misfolded proteins, while P56S-VAPB formed insoluble aggregates and lost the ability to mediate the pathway (loss-of-function), and suggested that P56S-VAPB promoted the aggregation of co-expressed wild-type (wt)-VAPB. In this study, a yeast inositol-auxotrophy assay has confirmed that P56S-VAPB is functionally a null mutant in vivo . The interaction between P56S-VAPB and wt-VAPB takes place with a high affinity through the major sperm protein domain in addition to the interaction through the C-terminal transmembrane domain. Consequently, wt-VAPB is speculated to preferentially interact with co-expressed P56S-VAPB, leading to the recruitment of wt-VAPB into cytosolic aggregates and the attenuation of its normal function. We have also found that expression of P56S-VAPB increases the vulnerability of NSC34 motoneuronal cells to ER stress-induced death. These results lead us to hypothesize that the total loss of VAPB function in unfolded protein response, induced by one P56S mutant allele, may contribute to the development of P56S-VAPB-induced amyotrophic lateral sclerosis.     

C-Terminal Auto-Regulatory Motif of Hepatitis C Virus NS5B Interacts with Human VAPB-MSP to Form a Dynamic Replication Complex

Hepatitis C virus (HCV) is a pathogen of global importance and nearly 200 million people are chronically infected with HCV. HCV is an enveloped single-stranded RNA virus, which is characteristic of the formation of the host membrane associated replication complex. Previous functional studies have already established that the human ER-anchored VAPB protein acts as a host factor to form a complex with HCV NS5A and NS5B, which may be established as a drug target. However, there is lacking of biophysical characterization of the structures and interfaces of the complex, partly due to the dynamic nature of the complex formation and dissociation, which is extensively involved in intrinsically-disordered domains. Here by an integrated use of domain dissection and NMR spectroscopy, for the first time we have successfully deciphered that the HCV NS5B utilizes its auto-regulatory C-linker to bind the VAPB-MSP domain to form a dynamic complex. This finding implies that the NS5B C-linker is capable of playing dual roles by a switch between the folded and disordered states. Interestingly, our previous and present studies together reveal that both HCV NS5A and NS5B bind to the MSP domains of the dimeric VAP with significantly overlapped interfaces and similar affinities. The identification that EphA2 and EphA5 bind to the MSP domain with higher affinity than EphA4 provides a biophysical basis for further exploring whether other than inducing ALS-like syndrome, the HCV infection might also trigger pathogenesis associated with signalling pathways mediated by EphA2 and EphA5.     

Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans

BACKGROUND: In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. RESULTS: Here, we present evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, we show that Galpha(s) signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. CONCLUSIONS: Regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells.     

Maintaining muscle mitochondria via transsynaptic signaling

Dominant VAPB mutations are implicated in neurodegenerative disease, including amyotrophic lateral sclerosis and spinal muscular atrophy. In the current issue, Han et al. (2012) uncover a mechanism through which the secreted VAPB MSP domain regulates actin organization and mitochondrial function in muscle cells through LAR and Robo receptor activation.     

Molecular cloning and partial characterization of a plant VAP33 homologue with a major sperm protein domain

In a search for proteins interacting with the resistance protein Cf9 from tomato, a new cDNA was cloned and characterized. Protein sequence database searches suggested that the 120 residue-N terminal domain of the encoded protein (named VAP27) is highly similar to the VAP33 protein family from animals, to uncharacterized plant proteins, and to a lower extent, to the major sperm protein (MSP) from nematodes. The second half of the protein is similar to VAMP and to the VAP33 N-terminus comprising a predicted coiled-coil region followed by a transmembrane segment. The sequence/structure comparison of VAP27 with the crystal structure of AsMSP1 from Ascaris suum, using molecular modeling with the threading method, suggested that the N-terminus of VAP27 does possess a MSP-like domain that might participate in the formation of a protein-protein network. The coiled-coil region of VAP27 was modeled based on the structure of the VAP- and VAMP-containing SNARE complex. The coiled-coil region might also be involved in protein-protein interactions similar to VAP-VAMP interactions.     

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.Subject terms: Mechanisms of disease, Amyotrophic lateral sclerosis